Production of 7-dehydrocholesterol



Patentod Feb. 17, 1948 UNITED STATES PATENT OFFICE PRODUCTION or'i-nnmaocnoms'rnaol.

No Drawing. Application January 24, 1945, Serial No. 574.446

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This invention relates to the production 01' compounds of the groupconsisting oi 'i-dehydro-cholesterol and its esters.

It is well known that certain 'I-delwdro sterols as for instanceY-dehydro-cholesteroi, ergosterol, etc. are valuable provitamins whichmay be antirachiticaliy activated to produce vitamin D.

7-dehydro-cholesterol may be obtained by chemical synthesis involvingthe thermal decomposition of 7-hydroxy-cholesterol or its carboxyb icacid esters. U. 8. Patents Nos. 2,098,984, 2,209,934 and others describethe production of 7-dehydro sterol compounds by the chemical removal ofthe elements of water or organic acid from 7-hydroxy-cholesteroi or itsesters. As a result of this reaction a double bond is formed between theseventh and eighth carbons in ring II of the sterol ring system which isconjugated with the existing double bond between carbons 5 and 6.

A serious disadvantage of this reaction is the formation of largequantities of various resinous and crystalline by-products, which uponsubsequent irradiation do not form products having antirachiticactivity. In the case of the provitamin derived from'7-hydroxy-cholesterol, the crystalline lay-product is generallyreferred to as lsodehydro-cholesterol, and has been identified in theliterature as A6:8-cholestadienol-3. (Windaus, Ann. 534, 22-41; Ch.Abst. 32, 4603.)

The following formulas further bring out the distinction:

CHI

7-dehydro-cholesterol (Si-isomer) BIC lsodehydro-cholesterol (EB-isomer)Hitherto no method has apparently been into a useful product, and thisby-product therefore constituted waste material.

This invention accordingly has as an object the production of usefulprovitamin material from the aforementioned lsodehydro-cholesterol. Afurther object is the conversion of said byproduct into thecorresponding 7-dehydro-cholesterol. Other and further important objectsof this invention will appear as the description proceeds.

Now according to my invention. the above objects are achieved in aneconomic and practical manner by subjecting the said crystallinebyproduct to heat treatment. which results in its conversion into theuseful 5:? isomer. 1 have found that the simple expedient of heattreatment will cause isomerization of lsodehydro-cholesterol into thenormal provitamin D.

The heat treatment, is for practical purposes achieved best by heatingthe lsodehydro compound at temperatures between 150 and 300 C. Theheating may be effected in a high boiling organic diluent or solvent.for in tance a high boiling hydrocarbon. alcohol or amine: or it may beeffected in a low boiling organic solvent, for instance benzene, ii aclosed vessel is employed. Optionally, isomerization may also beachieved by heating the lsodehydro compound by itself until it fuses.and maintaining a, temperature between 150 and 300 C. until a testsample shows that isomerization has proceeded to a satisfactory degree.

The by-product material being thus heattreated may be in the freehydroxy state, that is the 3-011 compound, or it may be in the form cl9, carboxyllc ester thereof, for instance the benzoate, acetate orpropionate. Such esters are often obtained directly in the process ofseparating the waste by-product from the principal reaction product inthe conversion of 'l-hydroxycholesterol diester into the corresponding'I-dehydro compound. Of course, common sense dictates that the esterselected for this purpose shall not be of an explosive or otherwisehazardous nature. It will be understood therefore that where I speak atrandom of esters of isodehydrocholesterol hereinafter in thisdisclosure. I am referring to non-hazardous esters, as would naturallybe selected for the process by a person skilled in the art.

When the lsodehydro material selected for treating is in the form of anester, there is a possibility that under certain conditions part of theester will decompose, liberating free organic known for transformingisodehydro-choiesterol as acid; and since acid has a tendency todeactivate dehydrosteroid compounds. it is recommended to add a mildalkali, such as sodium bicarbonate or an organic amine, in smallquantities to the isomerization mass. Alternatively, the organic solventmay be selected so as to create or maintain in the reaction mass a mildalkalinity, that is a pH of at least 7. From this viewpoint, liquidorganic bases which are inert toward the material being treated, areideal as solvent media. and may be illustrated by dimethyl-aniline,quinoline, pyridine or picoline, the reaction in the latter two casesbeing carried out in a closed vessel.

My invention, however, is by no means limited to the mentioned solventmedia, since excellent results are, according to my experience, obtainedalso with non-basic, inert solvents, such as terpineol, tetralin, benzylalcohol, benzylbenzoate, isophorone, petroleum hydrocarbons boiling inthe neighborhood of 200 C., and in general any inert, organic liquidboiling within the range of 150 to 300 C., and such heating may becarried out in the presence or absence of acid absorbing agents. Asalready said, the latter are not essential if the slight amount of aciddecomposition is not objectionable.

Where the thermal isomerization according to this invention has beenperformed on an ester of the isodehydro compound, the isomerized productmay then be subjected to saponiflcation in the customary manner, toliberate the tree 3-0H compound.

Without limiting my invention, the following examples are given toillustrate my preferred mode of operation. Parts mentioned are byweight. Example 1.-Isomerlzatlon of benzoate; terpineol,

as solvent 6.4 parts of isodehydro-chloesteryi benzoate (Windaus,Linsert and Eckhardt, Annalen 534, 22-41; melting range i38-140 C., withgreen fluorescence. clearing at 180 C.) were charged into a glass vesselequipped with thermometer, reflux condenser and agitator. 64 parts ofterpineol (B. P. 216-220 C.) were added and solution was effected byheating with agitation at IO-90 C. for 1 hour. 1.3 parts of sodiumbicarbonate were then added, and the mixture was heated to refluxtemperature and held at reflux for 2 hours. The charge was then cooledto 90 C., and 10 parts of water were added to dissolve soluble salts.The aqueous layer was removed and the washing operation was repeatedwith 10 parts of water. The mass was then distilled under vacuum toremove the bulk of the terpineol (55-58 parts), and 24 parts oi acetonewere added to the warm residue.

A crop of granular crystals precipitated, and these were filtered oilwith suction and washed with 1 to 2 parts of acetone. 5 parts of"I-dehydro-cholesteryl-benzoate were obtained, melting at 137-141 C.without green fluorescence, and clearing at 181 C.

To convert the above into the free 3-0H compound, thel-dehydro-cholesteryl benzoate was saponified by dissolving in parts ofbenzene, adding a solution of 1.3 parts potassium hydroxide in parts ofmethanol, and refluxing the mixture for 1 hour. 2.5 parts of water werethen added, and heating was continued until '7 to 8 parts of mixedsolvent were distilled oil. The residual mass was cooled to 10 C.,filtered under suction, and the crystalline residue was washed withmethyl alcohol. The product had a melting range oi -135 0., end an angleof rotation m=84', which properties agree well with those of crude'l-dehydro-cholesterol, as obtained in a typical deacylation o!'l-hydroxycholesteryl dibenzoate.

For analytical comparison, a sample of the isodehydrocholesterylbenzoate was subjected to saponification in the above manner withoutprevious isomerization. The resulting isodehydrocholesterol (tree OHiorm) had a melting range of 117-120" C. and m=--31.

Example 2.Isomerizatlon of the tree OH compound; dlmethyl aniline, assolvent The free 3-03 form of isodehydro-cholesterol is flrst preparedby charging 10 parts of the 3.5-

dinitrobenzoic ester of isodehydro-cholesteroi (M. P. 194 C.) into 20parts of benzene, adding 2 parts or potassium hydroxide dissolved in 32parts of methanol, and refluxing for 1 hour. The charge is then cooled,diluted with 100 parts of water, and the benzene layer is separated andwashed with water until free of soluble salts.

The benzene solution of isodehydro-cholesterol thus obtained is chargedinto a flask containing 30 parts of dimethyl aniline, and the benzene isdistilled off by heating to a. gentle reflux C.) over 2 to 3 hours. Thecharge is cooled slowly and then subjected to vacuum distillation toremove 25-28 parts of the dimethyl aniline. From the resultingconcentrated residue, 'l-dehydro-cholesterol may be recovered bycrystallization from a mixture of benzene and methanol.

For analytical purposes, or for the purpose of separating the 5:7 isomerfrom any residual 6:8 isomer, the crude reaction mass hereinaboveobtained may be converted into its 3.5-dinitro-- benzoic ester by thei'ollowing procedure: The concentrated residue hereinabove mentioned,after distilling oi! the bull: of dimethyl aniline, is diluted with 6parts of pyridine, and then "I parts of 3,5-dlnitrobenzoyi chloride,dissolved in 16 parts of dry benzene, are added. The mixture is allowedto stand for 2 hours and the ester of the isomerized sterol, whichcrystallizes out, is filtered off, and recrystallized from a mixture 01'equal parts of benzene and acetone. The yellow, crystalline product hasa melting range oi 209- 210 C., which corresponds to that of'l-dehydrocholesteryl-3,5-dinitrobenzoate.

From the mother liquors, some residual 6:8 isomer may be isolated as theflzfi-dinitro-benzoate by concentrating the liquors and filtering. Therecovered ester may then be saponifled to isodehydro-cholesterol asabove described, and may then be resubiected to isomerization. togetherwith an additional fresh portion oi the by-product. if desired. In thisfashion. by recycling repeatedly the recovered isodehydrocholesterol,the percentage conversion thereof into provitamin over a series oicycles can be made very high.

Example 3.Isomerization without alkali The procedure is essentially thesame as in Example I. For instance, 6 parts oi isodehydrocholesterylbenzoate are charged into a glass flask equipped with an agitator,reflux condenser and thermometer. Sixty parts of terpineol (boilingrange 216-220 C.) are added and the mass is heated slowly to a gentlereflux and held at reflux for 3 hours. Then, upon cooling to 90 C.,vacuum is applied and about 54 parts of the terpineol are distilledofl'. Into the remaining warm residue, 24 parts of acetone are added andthe granular crystals which form are filtered off and washed with 1 to 2parts of acetone. 'i-dehydro-cholesteryl benzoate is obtained whichmelts without fluorescence. This product may be saponified as in Example1, followed optionally by reesteriflcation with 3,5-dlnitrobenzoylchloride, as in Example 2 above, for the purpose of analytical study.

Example 4.Isomerizatin in the absence of a solvent Charge 5 parts oflsodehydro-cholesteryl benzoate into a glass vessel equipped with anagitator and a thermometer. Heat this material to a temperature of 210C., during which time. the melted crystals exhibit a green fluorescence.Hold the reaction temperature at 210 C. for 2 hours or until a sample ofthe reaction mass does not fluoresce on cooling to 130 C. This testindicates that the isomerlzation reaction is finished. Then cool to 80C. and add 6 parts of benzene, and 10 parts of acetone. Granularcrystals precipitate which are filtered at C., washed with 3 parts ofacetone and dried. From this crude product, 'I-dehydro-cholesterol inpure form may readi y be isolated by means of customary purificationsteps,

Example 5.-Isomerization of the benzoate; using glycerin as a diluentCharge 5 parts of isodehydro-cholesteryl benzoate, melting with bluishgreen fluorescence, into a glass flask equipped with an agitator,distilling condenser and thermometer. Add 25 parts of C. P. glycerin andheat the reaction mass to 210 C. During the course of this heating, abrilliant green and violet color display is observed. Hod the reactionmass at a temperature of 210 C. for 3 hours during which time the greenviolet color radually disappears, indicating that the isomerization hasbeen completed. If not agitated, glycerin separates from the moltensterol ester. Cool to 80 C., add parts of water and extract theisomerized sterol benzoate with 10 parts of benzene. Wash the separatedbenzene layer with 10 parts of warm water, separate and add 10 parts ofacetone to the benzene layer. A rop of granular crysta s precipitates,which after filtration, washing with 2 parts of acetone and dryingyields 'T-dehydro-cholesteryl benzoate, which melts withoutfluorescence. This product may be further purified to give pure7-dehydrocholesterol in good yield.

In all of the above examples, the isomerization ofisodehydro-cholesteryl benvoate to '7-dehydrocholesteryl benzoate isparticu arly characterized by the disappearance of the bluish greenfluorescence which is observed when isodehydro-cholesteryl benzoate isheated to its melting point. A comparison of the rotation in chloroformbetween samples of isodehydro-cholesterol and 7- dehydro-cholesterol,obtained by the saponiflcation of isomerized isodehydro-cholesterylbenzoate, shows that the alevo-rotetory angle has increased from -31 to84, The latter figure is in agreement with the rotation for7-dehydrocholesterol obtained from 7-hydroxy-cholesteryl dibenzoate bydebenzoylation followed by saponlfication. The melting ranges of thefree sterols before and after this isomerization also undergo acharacteristic change. The melting range increases from ll'I-120 C, toBil-135 C. which is closer to the melting range of crudeI-dehydrocholesterol, as above obtained. Additional proof of thechemical nature of the isomerized prod- 6 not is furnished byspectrographic analysis which shows that it possesses the characteristicabsorption lines of 7-dehydro-cholesterol.

In lieu of the 3-benzoate, other carboxylic acid esters of theisodehydro compound may be employed, which are not hazardous at thetemperature of isomerization. The ester is not limited to aromaticcarboxylic acids; the acetate and propionate are common example: of thealiphatic ester type. In addition, as already shown, the sterol may beisomerized also in free OH form.

The conditions of the reaction may be varied widely without departingfrom the scope of this invention. That is, the temperature used mayrange from to 300 C., but an increase in the time required for thecompletion of the isomerization is needed when operating at a lowtemperature. The solvents used in this reaction may be any organicmaterial which does not have an acid reaction (i. e, pH below '1) at thetemperature employed. Additional illustrations-of these may be found inthe numerous hydrocarbons, oxygenated compounds, and nitrogenous baseslisted in U. 8. Patents Nos. 2,255,815 and 2,341,733. Preferred solventsare those which are liquid at 200 C. A diluent which is not a solventfor isodehydro sterols may also be used. If a liquid boiling below 150C. is used, benzene for example, the preferred reaction temperature of200 C, may be attained by employing an appropriate pressure apparatus.

The addition of a mild alkaline reacting salt is a preferred embodimentof this invention but the lsomerization of isodehydro-cholesterol willtake place also by heating without the addition of such alkalinereacting substance. Some of the other alkaline reacting salts which maybe used are alkaline earth carbonates, bicarbonates, acetates, basicphosphates, etc.

Various amines have a beneficial efiect in this isomerizatlon and thisapplies both to isomerization in ester form and in free OH form. Someliquid bases, such as quinoline, pyridine, alpha picoline and others,are also useful in serving as an acid binding agent in the subsequentprocedure, if the crude isomerized sterol is to be purified byconversion into an ester thereof by the aid of an acyl chloride.

The uses and advantages 01' this invention will now be readily apparent.My invention makes it possible to reclaim an otherwise waste product inthe production of vitamin D into a useful product. Moreover, the usefulproduct thus obtained is identical with the principal product aimed atin the manufacture of provitamin from cholesterol, and thus adds to theoverall yield of the principal product. My process is also characterizedby extreme simplicity of procedure, requiring no complicated apparatusand no consumption of expensive chemicals and reagents, and beingefl'ected in essence by the simple expedient of heating.

The results of this invention are particularly surprising inasmuch asthe isodehydro-cholesterol used as starting material is itself formed ina thermal deacylation procedure which is often conducted in a reactionmedium and at a temperature similar to those used in this invention.

In the claims below where I speak of heating isodehydro-cholesterol oran ester thereof "in bulk," I mean by the phrase to designate treatmentof the above by-product material by itself, as distinguished iromheating in a solvent or diluent, and as further distinguished fromheating a reaction mass containing this sub- 7 stance in admixture with'I-dehydro-cholesteroi or an ester thereoi.

I claim as my invention:

1. The process 01' converting an isolated mass consisting essentially ofa compound selected from the group consisting of isodehydro-cholesteroland its esters into provitamin material, which comprises heating saidmass at a temperature between 150 and 300 C. to produce thecorresponding 'I-dehydro-cholesterol compound.

2. The process of converting an isolated mass consisting predominantlyof a compound seiected from the group consisting ofisodehydrccholesterol and its esters into the correspcnding7-dehydro-cholesterol compound, which comprises heating said mass to atemperature between 150 and 300 C. under mildly alkaline conditions.

3. The process of converting an isolated mass composed essentially of acompound selected from the group consisting of isodehydro-cholesteroland its esters into the corresponding 'l-dehydro-cholesterol compound,which comprises heating said mass to a temperature between 150 and 300'O. in an organic solvent in the presence of an acid absorbing agent.

4. The process of converting an isolated mass compound essentially of acompound selected from the group consisting of isodehydro-choiesteroland its esters into the corresponding 'l-dehydro-cholesteroi compound,which comprises heating said mass to a temperature between 150 and 300C. in the presence of a liquid nitrogenous base.

5. The process of producing 'l-dehydro-cholesterol, which comprisesheating isodehydrocholesterol in bulk at a temperature between 150' and300 C.

6. The process oi producing 'I-dehydro-choiesterol, which comprisesheating an ester 01 isodehydro-cholesterol in bulk at a temperaturemass.

JALIES ALLAN CALI-AN. REFERENCES CITED The following references are ofrecord in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,093,984 Windaus Nov. 16, 19372,209,934 Rosenberg July 30, 1940 2,255,815 Rosenberg Sept. 16, 19412,341,733 Meuly Feb. 15, 1944 OTHER REFERENCES Windaus, Annalen, vol.534, PM 22-41 (1933).

Certificate of Correction Patent No. 2,436,195.

February 17, 1948.

JAMES ALLAN CALLAN It is hereby certified that errors appear m thecorrection as follows:

column 7, line 28, for the word compoun at the said Letters Patentshould be read with numbered patent mring "sieve-rotatory"lasso-rotatory; first occurrence, read composed; and th thesecorrections therein that the same may con Patent Office.

rinted specification of the above lumn 5, line 64, for the word form tothe record of the case in the Signed and sealed this 27th day of April,A. D. 1948.

THOMAS F. MURPHY,

Assistant flommiuimr of Patents.

7 stance in admixture with 'I-dehydro-cholesteroi or an ester thereoi.

I claim as my invention:

1. The process 01' converting an isolated mass consisting essentially ofa compound selected from the group consisting of isodehydro-cholesteroland its esters into provitamin material, which comprises heating saidmass at a temperature between 150 and 300 C. to produce thecorresponding 'I-dehydro-cholesterol compound.

2. The process of converting an isolated mass consisting predominantlyof a compound seiected from the group consisting ofisodehydrccholesterol and its esters into the correspcnding7-dehydro-cholesterol compound, which comprises heating said mass to atemperature between 150 and 300 C. under mildly alkaline conditions.

3. The process of converting an isolated mass composed essentially of acompound selected from the group consisting of isodehydro-cholesteroland its esters into the corresponding 'l-dehydro-cholesterol compound,which comprises heating said mass to a temperature between 150 and 300'O. in an organic solvent in the presence of an acid absorbing agent.

4. The process of converting an isolated mass compound essentially of acompound selected from the group consisting of isodehydro-choiesteroland its esters into the corresponding 'l-dehydro-cholesteroi compound,which comprises heating said mass to a temperature between 150 and 300C. in the presence of a liquid nitrogenous base.

5. The process of producing 'l-dehydro-cholesterol, which comprisesheating isodehydrocholesterol in bulk at a temperature between 150' and300 C.

6. The process oi producing 'I-dehydro-choiesterol, which comprisesheating an ester 01 isodehydro-cholesterol in bulk at a temperaturemass.

JALIES ALLAN CALI-AN. REFERENCES CITED The following references are ofrecord in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,093,984 Windaus Nov. 16, 19372,209,934 Rosenberg July 30, 1940 2,255,815 Rosenberg Sept. 16, 19412,341,733 Meuly Feb. 15, 1944 OTHER REFERENCES Windaus, Annalen, vol.534, PM 22-41 (1933).

Certificate of Correction Patent No. 2,436,195.

February 17, 1948.

JAMES ALLAN CALLAN It is hereby certified that errors appear m thecorrection as follows:

column 7, line 28, for the word compoun at the said Letters Patentshould be read with numbered patent mring "sieve-rotatory"lasso-rotatory; first occurrence, read composed; and th thesecorrections therein that the same may con Patent Office.

rinted specification of the above lumn 5, line 64, for the word form tothe record of the case in the Signed and sealed this 27th day of April,A. D. 1948.

THOMAS F. MURPHY,

Assistant flommiuimr of Patents.

